Inflammation and DKK1-induced AKT activation contribute to endothelial dysfunction following NR2F2 loss.

NR2F2 is expressed in endothelial cells (ECs) and Nr2f2 knockout produces lethal cardiovascular defects. In humans, reduced NR2F2 expression is associated with cardiovascular diseases including congenital heart disease and atherosclerosis. Here, NR2F2 silencing in human primary ECs led to inflammation, endothelial-to-mesenchymal transition (EndMT), proliferation, hypermigration, apoptosis-resistance, and increased production of reactive oxygen species. These changes were associated with STAT and AKT activation along with increased production of DKK1. Co-silencing DKK1 and NR2F2 prevented NR2F2-loss-induced STAT and AKT activation and reversed EndMT. Serum DKK1 concentrations were elevated in patients with pulmonary arterial hypertension (PAH) and DKK1 was secreted by ECs in response to in vitro loss of either BMPR2 or CAV1, which are genetic defects associated with the development of PAH. In human primary ECs, NR2F2 suppressed DKK1, whereas its loss conversely induced DKK1 and disrupted endothelial homeostasis, promoting phenotypic abnormalities associated with pathologic vascular remodeling. Activating NR2F2 or blocking DKK1 may be useful therapeutic targets for treating chronic vascular diseases associated with EC dysfunction.NEW & NOTEWORTHY NR2F2 loss in the endothelial lining of blood vessels is associated with cardiovascular disease. Here, NR2F2-silenced human endothelial cells were inflammatory, proliferative, hypermigratory, and apoptosis-resistant with increased oxidant stress and endothelial-to-mesenchymal transition. DKK1 was induced in NR2F2-silenced endothelial cells, while co-silencing NR2F2 and DKK1 prevented NR2F2-loss-associated abnormalities in endothelial signaling and phenotype. Activating NR2F2 or blocking DKK1 may be useful therapeutic targets for treating vascular diseases associated with endothelial dysfunction.

Meta-analysis of blood genome-wide expression profiling studies in pulmonary arterial hypertension.

Inflammatory cell infiltrates are a prominent feature of aberrant vascular remodeling in pulmonary arterial hypertension (PAH), suggesting that immune effector cells contribute to disease progression. Genome-wide blood expression profiling studies have attempted to better define this inflammatory component of PAH pathobiology but have been hampered by small sample sizes, methodological differences, and very little gene-level reproducibility. The current meta-analysis (seven studies; 156 PAH patients/110 healthy controls) was performed to assess the comparability of data across studies and to possibly derive a generalizable transcriptomic signature. Idiopathic (IPAH) compared with disease-associated PAH (APAH) displayed highly similar expression profiles with no differentially expressed genes, even after substantially relaxing selection stringency. In contrast, using a false discovery rate of ≤1% and I2 < 40% (low-to-moderate heterogeneity across studies) both IPAH and APAH differed markedly from healthy controls with the combined PAH cohort yielding 1,269 differentially expressed, unique gene transcripts. Bioinformatic analyses, including gene-set enrichment, which uses all available data independent of gene selection thresholds, identified interferon, mammalian target of rapamycin/p70S6K, stress kinase, and Toll-like receptor signaling as enriched mechanisms within the PAH gene signature. Enriched biological functions and diseases included tumorigenesis, autoimmunity, antiviral response, and cell death consistent with prevailing theories of PAH pathogenesis. Although otherwise indistinguishable, APAH (predominantly PAH due to systemic sclerosis) had a somewhat stronger interferon profile than IPAH. Meta-analysis defined a robust and generalizable transcriptomic signature in the blood of PAH patients that can help inform the identification of biomarkers and therapeutic targets.

Pulmonary tumor thrombotic microangiopathy and pulmonary veno-occlusive disease in a woman with cervical cancer treated with cediranib and durvalumab.

BACKGROUND: Pulmonary tumor thrombotic microangiopathy (PTTM) is a rare cause of pulmonary hypertension that is associated with malignancies and is marked by the presence of non-occlusive tumor emboli and fibrocellular intimal proliferation of small pulmonary arteries leading to increased pulmonary vascular resistance and right heart failure. The diagnosis of PTTM is challenging to make pre-mortem and guidelines on treatment are lacking. CASE PRESENTATION: A 45-year-old woman with advanced squamous cell carcinoma of the cervix developed symptoms of dyspnea and evidence of right heart failure during a phase I clinical trial with cediranib and durvalumab. After an extensive evaluation, pre-capillary pulmonary hypertension was confirmed by right heart catheterization. Vasodilator therapy was initiated but resulted in the development of symptomatic hypoxemia and was discontinued. Despite continued supportive care, she continued to decline and was transitioned to hospice care. At autopsy, the cause of her right heart failure was found to be due to PTTM with features of pulmonary veno-occlusive disease (PVOD). CONCLUSION: PTTM and PVOD are important diagnoses to consider in patients with a malignancy and the development of right heart failure and may be manifestations of a spectrum of similar disease processes.

Raf/ERK drives the proliferative and invasive phenotype of BMPR2-silenced pulmonary artery endothelial cells.

A proliferative endothelial cell phenotype, inflammation, and pulmonary vascular remodeling are prominent features of pulmonary arterial hypertension (PAH). Bone morphogenetic protein type II receptor (BMPR2) loss-of-function is the most common cause of heritable PAH and has been closely linked to the formation of pathological plexiform lesions. Although some BMPR2 mutations leave ligand-dependent responses intact, the disruption of ligand-independent, noncanonical functions are universal among PAH-associated BMPR2 genotypes, but incompletely understood. This study examined the noncanonical signaling consequences of BMPR2 silencing in human pulmonary artery endothelial cells to identify potential therapeutic targets. BMPR2 siRNA silencing resulted in a proliferative, promigratory pulmonary artery endothelial cell phenotype and disruption of cytoskeletal architecture. Expression profiling closely reflected these phenotypic changes. Gene set enrichment and promoter analyses, as well as the differential expression of pathway components identified Ras/Raf/ERK signaling as an important consequence of BMPR2 silencing. Raf family members and ERK1/2 were constitutively activated after BMPR2 knockdown. Two Raf inhibitors, sorafenib and AZ628, and low-dose nintedanib, a triple receptor tyrosine kinase inhibitor upstream from Ras, reversed the abnormal proliferation and hypermotility of BMPR2 deficiency. Inhibition of dysregulated Ras/Raf/ERK signaling may be useful in reversing vascular remodeling in PAH.

Isolation of a circulating CD45-, CD34dim cell population and validation of their endothelial phenotype.

Accurately detecting circulating endothelial cells (CECs) is important since their enumeration has been proposed as a biomarker to measure injury to the vascular endothelium. However, there is no single methodology for determining CECs in blood, making comparison across studies difficult. Many methods for detecting CECs rely on characteristic cell surface markers and cell viability indicators, but lack secondary validation. Here, a CEC population in healthy adult human subjects was identified by flow cytometry as CD45-, CD34dim that is comparable to a previously described CD45-, CD31bright population. In addition, nuclear staining with 7-aminoactinomycin D (7-AAD) was employed as a standard technique to exclude dead cells. Unexpectedly, the CD45-, CD34dim, 7-AAD- CECs lacked surface detectable CD146, a commonly used marker of CECs. Furthermore, light microscopy revealed this cell population to be composed primarily of large cells without a clearly defined nucleus. Nevertheless, immunostains still demonstrated the presence of the lectin Ulex europaeus and von Willebrand factor. Ultramicro analytical immunochemistry assays for the endothelial cell proteins CD31, CD34, CD62E, CD105, CD141, CD144 and vWF indicated these cells possess an endothelial phenotype. However, only a small amount of RNA, which was mostly degraded, could be isolated from these cells. Thus the majority of CECs in healthy individuals as defined by CD45-, CD34dim, and 7-AAD- have shed their CD146 surface marker and are senescent cells without an identifiable nucleus and lacking RNA of sufficient quantity and quality for transcriptomal analysis. This study highlights the importance of secondary validation of CEC identification.

Washing older blood units before transfusion reduces plasma iron and improves outcomes in experimental canine pneumonia.

In a randomized controlled blinded trial, 2-year-old purpose-bred beagles (n = 24), with Staphylococcus aureus pneumonia, were exchanged-transfused with either 7- or 42-day-old washed or unwashed canine universal donor blood (80 mL/kg in 4 divided doses). Washing red cells (RBC) before transfusion had a significantly different effect on canine survival, multiple organ injury, plasma iron, and cell-free hemoglobin (CFH) levels depending on the age of stored blood (all, P < .05 for interactions). Washing older units of blood improved survival rates, shock score, lung injury, cardiac performance and liver function, and reduced levels of non-transferrin bound iron and plasma labile iron. In contrast, washing fresh blood worsened all these same clinical parameters and increased CFH levels. Our data indicate that transfusion of fresh blood, which results in less hemolysis, CFH, and iron release, is less toxic than transfusion of older blood in critically ill infected subjects. However, washing older blood prevented elevations in plasma circulating iron and improved survival and multiple organ injury in animals with an established pulmonary infection. Our data suggest that fresh blood should not be washed routinely because, in a setting of established infection, washed RBC are prone to release CFH and result in worsened clinical outcomes.

A unique population of effector memory lymphocytes identified by CD146 having a distinct immunophenotypic and genomic profile.

BACKGROUND: CD146 is a well described homotypic adhesion molecule found on endothelial cells and a limited number of other cell types. In cells from the peripheral circulation, CD146 has also been reported to be on activated lymphocytes in vitro and in vivo. The function associated with CD146 expression on lymphoid cells is unknown and very little information is available concerning the nature of CD146+ lymphocytes. In the current study, lymphocytes from healthy donors were characterized based upon the presence or absence of CD146 expression. RESULTS: CD146 was expressed on a low percentage of circulating T lymphocytes, B lymphocytes, and NK cells in healthy individuals. CD146 expression can be induced and upregulated in vitro on both B cells and T cells, but does not correlate with the expression of other markers of T cell activation. CD146 positive T cells do not represent clonal expansions as determined with the use of anti Vbeta reagents. Data suggest that CD146 positive cells have enhanced adherence to endothelial monolayers in vitro. Gene profiling and immunophenotyping studies between CD146+ and CD146- T cells revealed several striking genotypic distinctions such as the upregulation of IL-8 and phenotypic differences including the paucity of CCR7 and CD45RA among CD146 positive T cells, consistent with effector memory function. A number of genes involved in cell adhesion, signal transduction, and cell communication are dramatically upregulated in CD146+ T cells compared to CD146- T cells. CONCLUSION: CD146 appears to identify small, unique populations of T as well as B lymphocytes in the circulation. The T cells have immunophenotypic characteristics of effector memory lymphocytes. The characteristics of these CD146+ lymphocytes in the circulation, together with the known functions in cell adhesion of CD146 on endothelial cells, suggests that these lymphocytes may represent a small subpopulation of cells primed to adhere to the endothelium and possibly extravasate to sites of inflammation.

Detection of circulating endothelial cells and endothelial progenitor cells by flow cytometry.

The finding of angiogenic and vasculogenic cells in the peripheral circulation may have profound effects on the course of a variety of diseases ranging from cancer to cardiovascular disease. These cells are ascribed to be endothelial in nature and are generally referred to as circulating endothelial cells if mature or as endothelial progenitor cells if immature. Different approaches have been used to detect these cells, including in vitro culture, magnetic bead isolation, and flow cytometry. We review flow cytometric methods for the detection and enumeration of these cells and provide technical suggestions to promote the accurate enumeration of circulating endothelial cells and endothelial progenitor cells.